1. Tannin extracts and their BSA precipitation ability at different pH values in vitro
M. Schweigmann, F. Taube, M. Gierus
University of Kiel, Germany
Institute of Crop Science and Plant Breeding – Grass and Forage Science/ Organic Agriculture

2. Content Issue and hypothesis Tannins Extracts Material & Methods
Results & Discussion
Summary

3. Issue and hypothesis Issue:
dairy feeding: surplus of rumen degradable protein from grass = low nitrogen use efficiency (NUE)
options:
adding concentrates with low rumen degradability (soya)
adding secondary plant compounds (PPO, tannins)
Hypothesis:
Tannin extracts are suitable as feed additives to increase NUE.

4. Condensed tannins secondary plant compounds flavan-3-ol units
phenolic ring
condensed to oligomers, polymers
form complexes
reversible, pH-dependent reaction?
protect feed protein in the rumen (pH > 6), release again in the abomasum (pH < 3) = increasing feed protein use efficiency
5% CT to prevent bloat
(Haslam, 1996; Aerts et al., 1999; Barry & McNabb, 1999)

5. Material & Methods
tannin extracts from different sources: quebracho (heartwood): Schinopsis lorentzii mimosa (bark): Acacia mearnsii tara (pods): Caesalpinia spinosa gambier (leaves): Uncaria gambir
Table 1. Tannin extracts and their content of condensed tannins (CT) and total phenols (TP) in g kg–1
for comparison#
quebracho
mimosa
tara
gambier
birdsfoot trefoil
CT*
138
253 5 49 19
TP**
ca. 1000
967
772 48
* determined by butanol/HCl method (Terrill et al., 1992)
** determined by folin-ciocalteu method (Singleton and Rossi, 1965)
# masterthesis, same equipment (Hamacher, 2012)

6. Material & Methods
Precipitation: modulated method of Osborne & McNeill (2001)
bovine serum albumin (BSA), dissolved in a sodium-acetate-solution; 60µg per tube
pH values between 7/6 and 2
tannin extracts in amounts of 0 to 10mg per tube
Bio-Rad Protein Assay, photometrical measurement of colour reaction
log-logistic equation: f(x) = 0 + d / {1 + exp (b (log(x) – log(e)))}

7. Differences for extract plateau values
Table 2. Plateau values in µg BSA with statistical differences.
plateau
quebracho
mimosa
tara
gambier
pH 7
69,850aA
74,376abA
65,648aA
>100†aA
pH 6
64,779baA
81,902aB
70,432aA
>100†aAB
pH 5
57,489cbA
79,158acB
72,329aB
74,042aAB
pH 4
48,536cA
77,587abB
74,116aB
60,472aAB
pH 3
35,669dA
69,964bB
78,732aC
46,238aA
pH 2
25,125eA
71,079bcB
74,840aB
a,b: different small letters indicate significant differences between pH values within an extract (P<0.05)
A, B: different capital letters indicate significant differences between extracts at the same pH value (P<0.05)
†: out of range
Quebracho indicates potential for increased NUE of the dairy cow:
high precipitation at rumen pH; low precipitation at abomasum pH

8. Conclusions
Tannin source and pH value affected precipitation significantly.
Quebracho formed complexes at simulated rumen pH, but barely at simulated abomasum pH.
Precipitation profiles of mimosa, tara, and gambier did not support the given hypothesis.
next step: validating results using in vivo / in situ techniques;
testing reversibility of complexing in the abomasum  

9. Thank you very much for your attention!

10. References
Aerts, R.J., Barry, T.N., McNabb, W.C. (1999): Polyphenols and agriculture: beneficial effects of proanthocyanidins in forages. Agriculture, Ecosystems and Environment 75,
Barry, T.N. und McNabb, W.C. (1999): The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants. British Journal of Nutrition 81,
Hamacher, M. (2012): Sekundäre Pflanzeninhaltsstoffe und Proteinbindungsvermögen von Wiesenkräutern und Futterleguminosen. masterthesis at university of Kiel.
Haslam, E. (1996): Natural polyphenols (vegetable tannins) as drugs: Possible modes of action. Journal of Natural Production 59,
Osborne, N.J.T. und McNeill, D.M. (2001): Characterisation of Leucaena condensed tannins by size and protein precipitation capacity. Journal of the Science of Food and Agriculture 81,
Singleton, J. und Rossi, P. (1965): Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. American Journal of Enology and Viticulture 16,
Terrill, T.H., Rowan, A.M., Douglas, G.B., Barry, T.N. (1992): Determination of extractable and bound condensed tannin concentrations in forage plants, protein concentrate meals and cereal grains, Journal of the Science of Food and Agriculture 58,

11. Precipitation at pH 7 and 2
o quebracho ∆ mimosa + tara x gambier
Mimosa seemed to precipitate BSA most efficiently.

12. Differences for extract ED50 values
quebracho
mimosa
tara
gambier
pH 7
4,028aA
2,587aB
7,927aC
42,781aABC
pH 6
2,651bA
2,057bB
4,849bC
47,889aABC
pH 5
1,960adA
1,463cA
1,687cA
6,784aB
pH 4
10,623cA
0,596dB
0,733dB
3,289aC
pH 3
1,009dA
1,630bcB
1,354cA
3,862aC
pH 2
1,094abcdAB
3,256eA
4,029eB
41,390aAB
a,b: different small letters indicate significant differences between pH values within an extract (P<0.05)
A, B: different capital letters indicate significant differences between extracts at the same pH value (P<0.05)

13. Precipitation at pH 6 and 5
o quebracho ∆ mimosa + tara x gambier

14. Precipitation by quebracho
o pH2 ∆ pH3 + pH4 x pH5 ◊ pH6 pH7 ∆

15. Precipitation at pH 7 for CT

16. Precipitation at pH 6 and 2 for CT